Download Induction and Fixation of Polarity -Early Steps in Plant Morphogenesis

Develop. Growth & Differ., 34 (2), 115-125 (1992)
Development
Growth & Differentiation
Review
Induction and Fixation of Polarity
-Early Steps in Plant Morphogenesis
(polarity induction/polarity ftxation/rnicrotubules/plants/tropisrn)
Peter Nick’ and Masaki Furuya213
Frontier Research Program, Riken Institute, Hirosawa 2- 1, Wako-sht, Saitama 35 1-0I , Japan
able to survey the multitude of phenomena associated with polarity, a formal classification of polarity
is a necessary first step: We must ask, how are the
poles defined? How is a clear output guaranteed
under conditions of high informational noise? To
what extent does polarity really evolve de novo and
what is the role of external information? At what
level of complexity is polarity enscribed into the
organism? To what extent is the formation of
polarity reversible? How stable is polarity?
These are questions, to which, with our present
knowledge, we are still unable to provide a more
then a merely phenomenological answer. Nevertheless, such phenomenological answers might
help us to identify the best questions to be asked.
1. Introduction
Specific and complex forms of plants and
animals emerge from simple, very often symmetric,
origins. This process includes a chain of basic
events whereby two poles develop along an originally more or less homogeneous axis. Since the
eighteenth century such phenomena are referred
to by the term “polarity”. This concept simply
designates the specific orientation of activity in
space and involves no assumptions whatsoever as
to its causes (112). For example, growth gradients (41), the spatial order of cell divisions (28),
directed transport of hormones across tissue (91,
103, 104, 105) and structural or physiological differences between two ends of a cell (95) have
been described using this concept. The concept
of polarity is widely employed not only in theoretical
and experimental studies (32), but also in horiculture and agriculture (93). Polarity in animals has
been discussed extensively (23, 30, 36, 66, 69, 72,
106, 113, 122, 128). Polarity in plants has been
described in several reviews (9, 16, 54, 95, 103,
104, 105, 112).
Despite the accumulation of a considerable
amount of descriptive evidence, the molecular and
cellular mechanisms responsible for induction and
fixation of polarity have remained obscure. In
recent years, powerful molecular techniques have
been applied with increasing frequency in conjunction with the concepts established by classical
developmental biologists. At this stage, a review
of the classical concepts of polarity may be useful
for the design of future research. If we are to be
2. Brief synopsis of the induction of polarity in
animals
To construct a conceptional framework, it is
worth beginning with a brief consideration of the
induction of polarity in non-plant organisms.
Simple or complex polarit)/,
In cases of polarity in animal systems, one
pole might simply lack a property (Fig. l ) , that is
present at the other pole (simple polarity), or both
poles could be defined positively by a specific
property (complex polarity). Transplanation and
fusion experiments have shown two antiparallel
subpolarities for animal-vegetative polarity of sea
urchin eggs (1 1 , 47, 11 8) and amphibian embryos
A.
’
Current address lnstitut de Biologie Moleculaire des
Plantes, CNRS 12, rue du General Zirnrner, 67084 Strasbourg
Cedex, France
Current address Advanced Research Laboratory, Hitachi
Ltd , Hatoyama, Saitarna 351-03, Japan
To whom correspondence should be addressed (Tel & Fax
t-81-492-96-7511)
’
Simple
Polarity
Fig. 1
115
Complex
Polarity
Simple versus complex polarity
Copyright 0 by The Japanese Society of
Developmental Biologists 1992
All rights of reproduction In any form reserved
P. Nick and M. Furuya
116
(33, 46, 121), for prestalk-prespore polarity in Dictyostelium (1 27), and for head-foot polarity in Hydra (10). In Dictystelium (127), as well as in Hydra
(44, 75, 108),the substances involved in the induction of the subpolarities have been identified.
Genetic analyses of anteroposterior and dorsoventralpolarity in Drosophila have revealed complex polarities (2, 25, 26, 50, 62, 87). However,
the ventral and the anterior poles appear to be
necessary for the correct establishment of the
dorsal and the posterior poles. The dorsal and
posterior poles, on the other hand, are not required
for the correct establishment of the ventral and the
anterior poles.
B.
Graded or all-or-none polaritp
The difference between the poles (Fig. 2)
could be merely quantitative (gradedpolarity) but it
could also be qualitative (all-or-none polarity).
Gradients are usually found at the early stages of
establishment of polarity. Thus, foot and head
activators in Hydra (10, l08), the morphogens
involved in prestalk-prespore polarity in Dicfyostelium (127) and the products of maternal polarity
genes in Drosophila (2, 26, 62) are distributed in
gradients along the axis of polarity. However,
all-or-none polarities often prevail in establishment
of the final outcome. In Hydra (lO), Drosophila
(88), Dictyosfelium (127) and the sea urchin
embryo (22) the final organisms look fairly normal
even after artificial imbalance is introduced at early
stags. In Hydra, for instance, “partial heads” or
“partial feet” do not occur. However, the frequency, with which a head or a foot is formed depends
on the initial gradients (10). In such cases, socalled proportion regulation is observed, i.e., the
natural proportions are established even for severely imbalanced initial states. This phenomenon
implies a kind of decision-making mechanism that
transforms gradients of precursors into all-or-none
polarities.
Graded
Polarity
Fig. 2.
All-or-none
Polarity
Graded’versus all-or-none polarity.
C. Establishment of polarity: integration, redistribution or inheritance?
Polarity simultaneously implies both unity and
difference and there are two principal ways, by
which this dichotomy can be produced (34): Either
the poles are initially isolated (Fig. 3 ) and are later
linked by long-range signals (polarity by integration) or the unity precedes the difference, i.e.,
polarity appears as a result of the redistribution of a
uniformly distributed agent (polarity by redistribution). Polarity by integration is found in Hydra,
where locally restricted autocatalytic processes in
combination with long-range lateral inhibition bring
about foot-head polarity (30, 31). The long-range
interactions do not necessarily rely on actual inhibitors-simple competition of local sites for limiting
resources can fulfil the same function, as seen in
the establishment of prestalk-prespore polarity in
Dictyostelium (127). In many cases, however,
polarity is not really induced de novo, but derives
from a maternal polarity (polarity by inheritance).
The anteroposterior and dorsoventral polarities of
the Drosophila embryo depend on maternal
mRNAs, which are deposited during oogenesis by
the follicle of the mother (1, 2, 67). The polarity of
the egg is, therefore, based upon the polarity of the
follicle. This conclusion is supported by the
Inheritance
Fig. 3. Principal models for the establishment of
polarity.
117
Induction and Fixation of Polarity
effects of mutations that influence follicle shape:
they also interfere with the dorsoventral polarity of
the embryo (2). Impressive examples of polarity
by inheritance can be found in unicellular organisms, for example the flagella-stalk polarity in
Caulobacter crescentus (17, 110, 111) or the dorsoventral polarity of Paramecium (58). In these
cases, during each cell cycle, one pole is regenerated in what could be described as the “semiconservative replication” of polarity.
D. Tissue polarity, cell polarity or both?
Polarity can arise from a gradient accross the
tissue (Fig. 4), even if individual cells are not polar
(tissue polarity). Alternatively, the cells may be
polar but there may be no asymmetry at the level of
the whole tissue (cell polarity). Eventually, polar
cells may become arranged in a polar fashion
along the tissue. In multicellular nonplant organisms, tissue polarity appears to prevail: the determinants of anteroposterior polarity in the Drosophila oocyte are deposited in the poles in the form of
mRNA (65) and, after translation, the corresponding proteins, spread in gradients along the long
axis of the embryo. Secondary genes then become activated, depending on the levels of constituents of the primary gradients, with a resultant
coarse subdivision of the embryo even before
cellularization occurs (67, 87, 88). These areas
interact seqentially with one another and cause an
increasingly precise determination, down to stripes
of only one cell-width (51). In the end, the cells
themselves become polar, as shown by transplantation experiments with inverse transplants
(69, 70). However, cell polarity is secondary and
derives from tissue polarity in the manner of a
harmonic-equipotential system (23). Progressive
determination of the Bauplan down to the level of
individual cells by intercellular interactions seems
to be typical of animal development as found in
amphibia (47, 1 13, 116), nematodes (1 29), Drosophila (72, 97), sea urchins (76), Hydra (lO), and
Dictyostelium (1 27).
me’i
olarity
e
Fig. 4. Classification pf polarity with respect to the element that is polar.
Fig. 5. Stabilisation of the expression of polarity versus
the true fixation of polarity
118
P. Nick and M. Furuya
E. Fixation of polarity
After polarity has been established, it causes
developmental changes that can be long-lasting or
even irreversible. One might say that the manifestation of polarity has become stable (Fig. 5).
However, polarity could be already fixed before it is
expressed in a visible manner. In fact, in the
Drosophila embryo (2) and in amphibian eggs (29)
dorsoventral polarity becomes stable before it is
expressed. If eggs of Xenopus are tilted just at
the time when polarity fixation occurs, embryos
with two dorsal sides result. Establishment and
fixation of dorsoventral polarity appears to rely on
microtubules in the cortical plasma (24). A role
for the cytoskeleton in polarity fixation has also
been suggested for odontoblast polarity in the
mouse embryo (63), which depends on the presence of an actin-binding 169-kDa transmembrane
protein.
3.
Induction of polarity in plants
Transplantation experiments have provided a
useful approach to studies of the polarity in animals. However, they are difficult to carry out in
plants because the cell walls are rigid and the
vacuoles are large (3). Why is it then worth investigating plant polarity at all? There are two main
answers to this question: (i) plant cells are often
omnipotent and able to regenerate a whole organism (1 14), and it is certainly worth asking what
happens to cell polarity during this process; and (ii)
plants have to tune their morphogenesis to conform to their environmetn, - thus, induction of
polarity must be linked to transduction of external
stimuli.
The induction of polarity in plants has been
studied extensively in the following two systems:
(A) Thallus-rhizoid polarity in phaeophycean zygotes and pteridophytean spores (89, 95, 99, 100,
lOl), and (B) Shoot-root polarity in higher plants
(32, 102, 104, 120).
A. Induction of cell polarity: Thallus-rhizoid
poiarity
In the zygote of Fucus, a colourless rhizoid
appears at one pole and the plastids migrate into
the opposite thallus pole. The direction of this
polarity can be influenced by a range of environmental factors, such as light, temperature, an electric field or a chemical gradient (101, 124). The
effects of light are due to as yet unidentified
blue-light receptors (7, 40). However, in certain
species of ferns and mosses, induction of polarity
can be triggered by a membrane-bound form of
the biliprotein phytochrome, which absorbs in the
red region of the visible spectrum (56). The environmental gradients that induce polarity can be
very small and the system is fairly resistant to
noise, suggesting extensive signal-amplification
mechanisms (30, 39). Sensitivity can become so
high that, even in a putatively symmetric environment, stochastic fluctuations can act as polarizing
stimuli (8). As demonstrated with the spores of
Equiseturn, a variation in the strength of the inducing gradient does not affect the expression of
polarity in the individual cell. However, it affects'
the frequency, within the population, with which
polarity is aligned correctly (39). This phenomenon resembles the situation in Hydra and is
consistent with the idea of an all-or-none polarity.
Irradiation with strong polarized light can cause
doubling of the rhizoid pole (53),which suggests
that polarity is induced de novo and is not inherited. The mechanism of induction of polarity has
been partially clarified for the Peivetia zygote (99,
100). Polarity develops parallel to gradients of
calcium, sodium and potassium ions, which were
measured by a very elegant technique (57): the
cells were sucked onto a fine nickel mesh and,
thus, served as insulators of the chambers above
and below the mesh. The ionic composition of the
chambers could be changed and currents measured with a vibration electrode. The future rhizoid pole exhibited an increased influx of calcium
ions into the cell, whereas the opposite pole was
characterized by a decreased influx of calcium
ions. For stimulation by an external gradient of
calcium ions, these data imply redistribution of
calcium pumps towards the site at which the
external concentration of calcium ions is minimal
(i.e., the future rhizoid pole). Similar autocatalytic
"self-electrophoresis" (55) was found in green
algae and mosses (124) and it appears to be a
general requirement for induction of polarity by
various environmental stimuli (98). Thus, thallusrhizoid polarity evolves by redistribution. How it is
transduced into the final form and whether and how
it is fixed, remain questions that must be answered
Polar transport of ribosomes (94), polar activation
of calmodulin and, as a consequence, the polar
phosphorylation of proteins (13) and directed
changes in the cytoskeleton (60) are some of the
phenomena that have been discussed in this context.
Induction and Fixation of Polarity
B. Induction of polality in multicellular plants:
Shoot-root polarity
Because of the horticultural importance of
polarity in grafting (93), polarity was already the
focus of scientific interest many years ago. Polarity was detected as a basoapical gradient in the
capacity of root stocks to regenerate adventitious
shoots. In addition, there is an antiparallel gradient in the capacity of the scion to regenerate
adventitious roots (32). The earliest theory on
shoot-root polarity was formulated by Marquise
Duharnel du Monceau in the eighteenth century.
He postulated the existence of two morphogenetic
factors, a heavy “root sap” and a light “shoot sap”,
which were directed by gravity towards the respective poles, accumulated there and triggered the
formation of roots and shoots, respectively (74).
In fact, the existence of such morphogenetic factors and their transport in the phloem was demonstrated in elegant incision experiments (35). The
question then arose as to whether this polarity
could be oriented by gravity. Early experiments
with inverted sunflower seedlings by gravity. Early experiments with inverted sunflower seedlings
yielded positive results (113), but it was not possible to reverse polarity in adult plants. In a famous
experiment, an inverted segment of a Salix branch
regenerated roots and shoots according to the
original polarity, and not according to its actual
orientation (120). By contrast, in rhizome cuttings
of Cordyljne, incubated horizontally, a new polarity
was inducible perpendicular to the original polarity,
although the latter still persisted and, as a result, a
quatripolar situation developed (32). In the
siphonal alga Caulerpa, polarity could be inverted
easily even by light (86). Depending on their
experimental material, researchers either favoured
the hypothesis of Sachs (102) that polarity is induced de novo or the opposing view, formulated
by Vochting (120), that polarity is inhertied and
stable. A synthesis was attempted by Goebel
(32), who showed, in elaborate cutting and regeneration studies, that an apicobasal flux of an unknown substance defines shoot-root polarity. If
this flux is interrupted or inverted, locally restricted
inversion of polarity can occur. This flux, in recent
years, has been shown to be that of auxin (103,
104, 105). Vessel regeneration in Coleus stems,
as directed by shoot-root polarity, was mainipulated in a predictable way by a series of ingenious
experiments in which incision was combined with
local application of auxin (104, 105). The
mechanism of induction, again, involves autocatalytic cycles: if within an initially homogeneous
119
distribution of auxin across the (still parenchyrnatic) tissue, the auxin flux is increased locally (for
instance, by blocking other drainage paths), the
increase leads to accelerated differentiation of
vessels at this site. Since those developing vessels can already transport more auxin per unit time,
they will deplete the neighbouring areas of auxin.
A few vessels will form and mutually compete for
auxin. With time, the vessels differentiate progressively, cortical microtubules are arranged in
transverse rings and, eventually, lignification leads
to some kind of stabilisation (104, 105). Strictly
speaking, it is the expression of polarity that becomes stable, not the polarity itself (103).
Separation of the two phenomena was emphasized
by Vochting and illustrated by an experiment with
dandelion roots (Fig. 6): a root segment was buried
upside-down after the shoot pole had been sealed
with resin. Under these circumstances, adventitious shoots formed at the (now upper) root pole.
This formation of shoots continued for several
months, and the original polarity seemed to be
reversed because shoots emerged preferentially at
the former root pole. However, when a segment
was cut from this inverted plant and buried without
sealing of the poles, the original polarity was clearly expressed and adventitious shoots emerged
exclusively at the original shoot pole. Thus, the
original polarity persisted unaltered even though its
manifestation had remained latent for several
months.
A precursor of shoot-root polarity can be detected already in the zygote: The zygote displays a
distinct cell polarity with a vacuolar pole at the
micropylar site (the future suspensor or root pole)
and an opposed cytoplasmic pole (1 14). It
appears that the cytoplasmic pole exerts an inhibitory effect upon the suspensor pole, which can be
removed by microsurgery or loss-of-function mutations (71). Differentiation is initiated from the cytoplasmic, embryonic pole (73, 96, 109, 114, 123).
It is unknown, whether or not the maternal tissue
imprints its polarity upon the embryo sack. Maternal mRNAs, as in case of the Drosophila embryo,
appear, however, to play a minor role (71). The
role of auxin fluxes in the early establishment of
shoot-root polarity remains to be defined. It is
clear, however, that the apical meristem does not
have the same impact on embryonic polarity as it
has during postembryonic development (1 14). In
the vegetative shoot, the polarity, although maintained by auxin fluxes, originates from continuous
and unequal cell divisions in the meristem initials
(114). This process can be disturbed by simple
120
P. Nick and M. Furuya
ORIGINAL
POLARITY
Fig. 6.
INVERTED POLARITY
EXPRESSION
Schematic representation of Vochting's experiment with Taraxacum
treatments that lead to symmetric divisions of
meristem cells (5). Nevertheless, the basis of
polarity in meristem cells (which is the ultimate
cause of shoot-root polarity) remains obscure.
From this initial cell polarity, tissue polarity
emerges as a secondary phenomenon, as can be
concluded from the capacity of apical fragments
for conspicuous proportion regulation (4, 92, 115).
In these respect, their behaviour resembles that of
split amphibian embryos (113). Interactions between different apical regions eventually determine
the plant pattern, as shown by microsurgery (64,
92, 114) and the analysis of pattern mutants (59,
68, 71). Thus, determination proceeds from the
cellular to the tissue level, which, in a sense, is the
mirror image of the harmonic-equipotential system
in animals (22).
4.
ORIGINAL
POLARITY
Polarity and tropism
Polarity in the unicellular zygote of Fucus can
be manipulated easily by environmental factors.
Shoot-root polarity of higher plants is not as readily
accessible. However, it does occur in multicellular organisms in which intercellular interactions can
be studied. Combining the advantages of both
types of systems is warranted. Although they
have not been on the mainstream of research on
plant morphogenesis, photo- and gravitropism provide appropriate systems with which to examine
such a combination. In fact, tropism and the
induction of polarity share many characteristic
traits: autocatalytic signal amplification (42),
efficient elimination of stochastic noise (30, 42),
induction by as yet unidentified blue-light receptors (7, 21, 40) and the importance of auxin (91,
103, 104, 105). Thus, it is not astonishing that
polarity has been described as a tropistic signal
chain which is extended by a fixation step (42).
Moreover, tropism, as well as the induction of
polarity, cannot be explained in terms of responses
of isolated cells, but they imply interactions between cells. This "holistic" nature of tropism has
been demonstrated repeatedly, for example, in
balancing experiments with the sporangiophore of
Phycomyces (271, by the observation that auxin
and growth are redistributed across photo- or
gravitropically stimulated grass coleoptiles (14, 19,
48, 49, 90, 91, 125, 126), by the failure to induce a
red-light tropism in coleoptiles, even though a
gradient of red light is detected by the plant (45),
and by the finding that the coleoptile recognizes
the direction of light by measuring the gradient of
light across the tissue (15, 48, 61). Phototropism
appears to induce a tissue polarity (15), which is
then expressed as the transverse transport of auxin
(19, 91, 125, 126). This phenomenon is remarkable because gravitropism must derive from some
sort of cell polarity (77). The gradient of gravitational acceleration across the coleoptile is certainly
too small to be sensed. As for shoot-root polarity
(32, 120) tropistic polarity can be separated from
Induction and Fixation of Polarity
its expression as curvature: if curvature is suppressed by cold treatment (20), decapitation (12) or by
specific inhibitors of plasma membrane ion-pumps
(37, 38), a long-lasting memory of the experienced
tropistic stimulation is, nevertheless, retained.
This tropistic "mneme" (12, 20) or "memory" (37,
38) persists for many hours and is expressed
readily as curvature if the suppression is removed.
The similarity to the experiment with segments of
dandelion roots (120) is evident (Fig. 6).
In maize coleoptiles, such a spatial "memory"
can be induced even by a unilateral pulse of blue
light and be rendered visible even ten hours later
as strong and stable curvature in the direction of
the light pulse by transferring the plants to a
horizontal clinostat (such that gravity acts symmetrically). This light-induced stable transverse
polarity derives from a labile precursor and is fixed
about two hours after induction (78, 85). It is
expressed in an ail-or-none fashion. Incomplete
polarity could not be produced even by varying the
strength of the applied stimuli (85). By analogy to
head-foot polarity in Hydra (1 0) and thallus-rhizoid
polarity in Fucus (37) variations in the strength of
the inducing gradients are mirrored by the frequency of plants where stable polarity is manifest.
Although transverse polarity is of the all-or-none
type, its early precursors can be shown to be
distributed in a gradient (85). It should be mentioned that a similar polarity can be induced by
gravity (78) and is expressed by an all-or-none
pattern as well (82). However, the respective
transduction chain is clearly separate from the
blue-light-induced transverse polarity (43, 82, 119).
By analogy to the double rhizoids in Fucus (53), a
stable symmetry could be induced if the opposing
light pulse was given exactly at the time at which
polarity fixation takes place (78, 85). This
observation indicates the de novo induction of
blue-lig ht-ind uced transverse polarity, consistent
with the old theory of Sachs (102). It should be
mentioned, however, that inheritance of polarity
according to Vochting (120) exists in the case of
the dorsoventral polarity that causes nastic curvature (79), and it interacts with transverse polarity in
an all-or-none fashion. Although triggered by
blue light, transverse polarity relies on a signal
chain, which is clearly separate from events that
mediate phototropism, as shown by time-course
and fluence-response studies (85). The separation occurs before the evolution of the phototropic
tissue polarity (82, 85).
Cortical microtubules in the outer epidermis
respond to light and gravity with a resultant orienta-
121
tion gradient across the coleoptile (81). Although
correlated in terms of timing and direction with
tropistic bending, the phenomena can be distinguished by the means .of microtubule-eliminating
drugs (83) and rotation on a clinostat (84).
However, microtubules exhibit a much closer involvement in the light-induced transverse polarity
(80), and a fixation of the orientation of microtubules occurs about two hours after a unilateral
blue-light pulse, at the same time as the fixation of
transverse polarity. This fixation is brought about
by a loss of microtubule motility and extends to
both poles. Thus, both poles of transverse polarity are positively defined (complexpolarity). Intact
actin microfilaments appear to be essential for
polarity fixation (80). A conspicuous cell polarity
can be observed in the case of reorientation of
microtubules, since only microtubules adjacent to
the outer epidermal wall appear to be responsive
(52, 81). The significance of a cell polarity is
evident from the fact that both gravity- and bluelight-induced transverse polarities can be reversed
by symmetric irradiation with blue light (85, 107).
5. Summary and prospects
Our knowledge of the induction of polarity in
plants, although still fragmentary, already allows us
to make certain generalizations:
1. There is a significant body of evidence for
extensive signal amplification that leads to all-ornone decisions. Where the underlying mechanism could be uncovered, locally restricted autocatalytic processe, in combination with long-ranging
inhibitory interactions, have been found to be important. In contrast to the situation in animals, this
long-ranging inhibition is produced by mutual competition for limiting resources rather than by actual
inhibitors.
2. Whereas polarity in animals seems to be complex for the most part, the situation in plants is less
unequivocal: thallus-rhizoid polarity in Fucus
appears to be simple; light-induced transverse
polarity in maize coleoptiles is complex. However, the graminean coleoptile might be a special
case because its lifespan is determined and cell
divisions do not occur during later phases.
Usually, plants are characterized by "open" organization that involves a high degree of flexibility.
This requirement might be more easily met by a
simple pohrity, which is less difficult to reorient.
3. Pokrity fixation appears to occur concomitantly with (shoot-root polarity) or later (light-induced
transverse polarity) than the expression of polarity.
122
P. Nick and M. Furuya
It is possible, however, to distinguish between
expression and fixation by preventing the expression of polarity and restoring it after some time with
an examination of whether or not the underlying
polarity had remained stable. A role for the cytoskeleton, and for microtubules in particular, in the
expression of polarity and its fixation has been
assumed for both thallus-rhizoid polarity and shootroot polarity. Such a role could be demonstrated
in the light-induced transverse polarity of maize
coleoptiles. Thus, stimulus-induced reorientations of microtubules and their membrane interactions play an important role.
Although some formal characterization of the
induction of polarity and its fixation in plants
appears to be possible, most of the mechanisms
involved remain obscure. It is not known how
some of the inducing stimuli are perceived, nor are
the signals known by which the individual cells
communicate in order to bring about an ordered
response. The cytoskeleton is very likely to be a
mediator for polarity. At least it can serve as a
marker of cell polarity. However, it is still unclear
by what mechanisms the cytoskeleton can be
restructured, how such structural changes are
directed, and how the cytoskeleton interacts with
the plasma membrane. Many of these questions
were, in fact, already posed a long time ago, when
they could not be answered, because the necessary tools were not available. Some of these tools
are now provided as a result of recent achievements in molecular biology and biochemistry. A
successful combination of old questions and new
tools must take into account the fact that polarity is
based on spatial order. Thus, assays in situ,
capable of distinguishing the responses of individual cells are warranted.
Acknowledgments: A fellowship from the Science
and Technology Agency Japan to P. N. is gratefully
acknowledged. The authors thank Dr. E. Lopez for
reading the manuscript.
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